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Biochemistry

Biochemistry. Dept. of Biochemistry and Molecular Biology. Professor Wu Yaosheng 2009-11. 小船上的人定是不知道，自己竟也成了美景的一部分。. 发现决定人类语言功能关键基因. 为什么人能说话而其生物学近亲黑猩猩却不能？英国 《 自然 》 杂志 11 月 12 日刊登研究报告说，答案可能就在基因 FOXP2 上，这个基因的人类版本与黑猩猩版本仅有两点小小的不同，但却因此赋予人类独特的语言能力。

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Biochemistry

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  1. Biochemistry Dept. of Biochemistry and Molecular Biology Professor Wu Yaosheng 2009-11

  2. 小船上的人定是不知道，自己竟也成了美景的一部分。小船上的人定是不知道，自己竟也成了美景的一部分。

  3. 发现决定人类语言功能关键基因 为什么人能说话而其生物学近亲黑猩猩却不能？英国《自然》杂志11月12日刊登研究报告说，答案可能就在基因FOXP2上，这个基因的人类版本与黑猩猩版本仅有两点小小的不同，但却因此赋予人类独特的语言能力。 美国加利福尼亚大学等机构的研究人员报告说，他们发现FOXP2基因在人类语言功能形成过程中发挥着核心作用。这个基因会指导合成一种特殊蛋白质，这种蛋白质又会与DNA（脱氧核糖核酸）结合，对其他基因的功能造成影响。因此，虽然实验显示这个基因的人类版本与黑猩猩版本只有两处氨基酸不同，但在同样的培养环境下，该基因的人类版本会增强61个基因的作用，同时抑制另外51个基因的作用。 在这些受影响的基因中，一些与大脑发育有关，FOXP2基因可以通过它们影响大脑中的语言功能区域和神经网络。另一些受影响的基因与咽喉部位的软组织发育有关，FOXP2基因可以通过它们来影响与语言功能有关的器官结构

  4. 我国科学家首提“人类泛基因组” 人类基因组存在着种群特异甚至个体独有的DNA序列和功能基因 经过不懈研究和攻关，我国科研人员在人类基因组研究中获得新的重大进展――发现人类基因组中存在着种群特异甚至个体独有的DNA序列和功能基因。科研人员还首次提出了“人类泛基因组”的概念。 由深圳华大基因研究院领衔，华南理工大学参与的研究论文《构建人类泛基因组序列图谱》12月7日在国际著名科学期刊《自然—生物技术》（Nature Biotechnology）上发表。 在研究中，科研人员运用第二代测序技术和自主研发的基因组组装工具，对“炎黄一号”基因组――首个亚洲人个人基因组进行了进一步的深度测序和拼接，发现人类基因组中除原先公认的单核甘酸多态性、插入删除多态性和结构性变异以外，还存在着种群特异甚至个体独有的DNA序列和功能基因，例如主要在亚洲人群内特有的基因序列。

  5. 我国科学家首提“人类泛基因组” 人类基因组存在着种群特异甚至个体独有的DNA序列和功能基因 科研人员同时对近两年发表的非洲人基因组和韩国人基因组进行了重新组装，也得到类似结论。科研人员还首次提出了“人类泛基因组”的概念，即人类群体基因序列的总和。 国际人类基因计划基于欧洲人DNA完成的参考基因组序列，是目前绝大多数人类基因组学研究的数据基础。多年来，大多数科学研究都认为每个个体的基因组均与这一参考基因组相似，仅有替换或重排性质的变化。 专家指出，这一研究树立了新的人类基因组测序标准，进一步证明自主构建中国人群医学基因组学图谱、推进个人基因组研究和个体化医学研究的必要性，是中国科学家在人类基因组研究领域的又一重要贡献。 在论文同行匿名审稿过程中，一名科学家评价说：“这是一篇激动人心，发人深思，严谨清晰的文章。除了对新序列的检出和分类，这篇文章还通过使用相当有趣的独创的分析方法，增强了我们对这些新序列中所能展示的种群多样性和进化保守性的认识。”

  6. Chapter 11 RNA Biosynthesis

  7. Two kinds of RNA biosynthesis in organisms: Transcription RNA replication 7

  8. Transcription----to synthesize RNA with DNA single strand as template Introduction 1. Transcription is the first stage of the process of gene expression. 2. Transcription processes have to suffer strictly regulation to meet the needof development, morphogenesis and physiological functions of organisms . 3. The products of transcription are RNA 8

  9. General picture of transcription 9

  10. Main Contains ★ Overview of Transcription ★ Transcription in Eukaryotes ★ Processing of Eukaryotic RNA ★ Transcription in Prokaryotes ★ RNA Dependent RNA Synthesis 10

  11. Key Points ★ Transcription characteristics ★ Transcription system ★ RNA polymerases ★ Transcription initiation ★ Postranscription modification 11

  12. Section One Overview of Transcription

  13. RNA DNA Transcription The process which RNA polymerase catalyzes the yield of RNA (tRNA, mRNA, rRNA ) with one of double strands of DNA as template, NTPs as precursors, in the light of the rule of complementary base pairing. 13

  14. The requirements of transcription Precursors : NTP (ATP, UTP, GTP, CTP) Template:DNA （one strand ) Enzyme:RNA polymerase, RNA-pol Other proteins (transcriptional factors et al ) 14

  15. Comparison of Replication and Transcription 15

  16. 1. General characters of transcription • Only one strand of DNA molecule serves as template • Asymmetric transcription • No primer needed • Initiation at promoter site 16

  17. DNA Transcription plot 5’……GCAGTACATGTC…………3’ 3’……c g t c a t g t a c a g…………5’ DNA transcription 5’……GCAGUACAUGUC………3’ mRNA translation N…... Ala Val His Val.. ………….C peptide Note：capital letters means the code strand, small letters means the template strand 17

  18. Some important concepts DNA template Template strand, antisense strand, Watson strand Coding strand, sense strand, Crick strand Structural gene Asymmetric transcription 18

  19. Direction of transcription: 5’ →3’ Direction of template reading: 3’ →5’ 19

  20. Asymmetry transcription 5’ 5’ 5’ 3’ 5’ 3’ 5’ Structure gene Template strand Coding strand Arrowhead means the direction of transcription 20

  21. For asymmetric transcription, there are two meanings: (1) Only one strand of a gene can serve as template, the other which is complimentary to the template strand can’t be transcripted. (2) Not all the template strands of genes are found in the same strand on DNA molecule. 21

  22. 2. DNA Dependent RNA Polymerase The enzyme related to transcription is RNA polymerase, which is termed as DNA dependent RNA polymerase (DDRP) or RNA pol, or transcriptase. It catalyzes the following reaction: DNA template (NTP)n pppN(pN)n-1 + (n-1)PPi RNA pol Mg2+, Zn2+ 22

  23. DNA template strand DNA template strand 5` direction 3` OH OH U U OH OH Transcription by RNA polymerase. In each step the incoming ribonucleotide selected is that which can base-pair with the next base of the DNA template strand. 23

  24. A 3`-5`phosphodiester bond is formed, extending the RNA chain by one nucleotide, and pyrophosphate is released. Overall the RNA molecule grows in a 5`3`direction. 24

  25. Eukaryotic RNA polymerase Amanitin，鹅膏蕈碱；refampicin, 利福平 25

  26. Prokaryotic RNA polymerase ( E. coli) 26

  27.         ω  ω RNA polymerase in prokaryotes only one RNA pol has been found: α2ββ’ωσ(holoenzyme ) α2ββ’ω( core enzyme ) Holoenzyme Core enzyme 27

  28. Core enzyme: theRNA polymerase without the  subunit is called core enzyme (2 ω). Holoenzyme:It caninitiate transcription specifically at promoter sites and catalyze polymerization of two free NTPs. Core enzyme: It can notinitiate transcription specifically at promoter sites but can catalyze RNA elongation. 28

  29. There are more than one kind of  factor in prokaryotes  70carries out the promoter recognition process on their own, mainly responsible for thehousekeeping geneexpression. ‘Housekeeping’ genes are those that encode many proteins needed for routine cell functions and which are therefor expressed at low rates in all cells.  32is responsible for theheat-shock geneexpression under some emergent cases. 29

  30. ω RNA polymerase binds to DNA at the Transcriptional start point How many kinds of RNA polymerase have been found in prokaryotes or in eukaryotes? 30

  31. Questions 1. The RNA polymerases that transcribe bacterial DNA or eukaryotic nuclear DNA are A. multisubunit enzymes and partially homologous. B. monomeric and very large. C. multimeric and interchangeable. D. only active inside the cell. E. highly glycosylated in their active forms. 31

  32. Questions 2. RNA chain elongation can be inhibited by A. Repressors. B. Rifampicin (a Rifamycin derivative). C. Actinomycin D. D. RNases. E. 0.5 M NaCl. 32

  33. Section Two Transcription in Eukaryotes

  34. The process of transcription is generally divided into three steps: Initiation Elongation Termination 34

  35. Requirements of transcription initiation RNA pol, promoter, TFs, precursors (NTP) Transcription factors and promoters 35

  36. Rbp2 Rbp7 Rbp6 Rbp1 Rbp5 Rbp4 Rbp11 Rbp12 Rbp3 Rbp8 Rbp10 Rbp9 1. Synthesis of mRNA in Eukaryotes RNA polymerase II is responsible for biosynthesis of mRNA in eukaryotes It contains 12 subunits: The largest one contains carboxyl-terminal domain, CTD RNA pol II 36

  37. Structural gene Promoter AATAA GC TATA CAAT intron -110bp -25bp - 40bp exon Enhancer Modification site Initiation site Real termination site Structures of gene in eukaryotes: Promoter, cis-action elements, exon, intron (A point for cutting and adding a tail ) 37

  38. enhancer P E1 I1 E2 I2 E3 +1 -110 -25 - 40 5’ T 3’ CAAT box GC box YYAN YY TATA box 3’ A 5’ Upstream elements BRE Inr Genes in eukaryotes are split gene (断裂基因） A gene consists of exons and introns. Promoter structure: RNA pol II Class two of promoter TF II BRE (TF II Brecognition element ); Inr (initiator) 38

  39. Class and functions of TF II 39

  40. 1.1 Initiation of mRNA Synthesis ◆To form pre-transcription initiation complex(PIC) First, a closed complex (闭合复合物），Secondly, an open complex ( 开放复合物） ◆To form a transcription bubble (转录泡)----transcription initiation complex ◆To join two nucleotides to form pppGpN-OH with first phosphodiester bond 40

  41. The forming of transcription initiation complex (1) TFIID binds to the TATA box. The key subunit of TF II D is TBP(TATA box-binding protein). (2) TFII A binds, followed by TF II B. (3) RNA polymerase II, which has already complex with TFIIF,binds followed by the binding of TFIIE,H. (4)The transcription begins when forming oftranscription initiation complex. (5)It is a basal transcription apparatus. Transcription is only at a low rate. For a high rate of transcription, other transcription factors are required. 41

  42. TATA TATA TATA TATA A A E H Initiation of transcription by RNA polymerase II.TFIIDbinds to the TATA box followed in order by the binding ofTFIIA,TFIIBand a pre-formed complex ofTFIIF-RNA polymerase II. SubsequentlyTFIIE, TFIIH bind in order and transcription then starts about 25 bp downstream from the TATA box. TF II D D TFIIA, TFIIB, TFIIF, RNA polymerase II D Pol II B F Note that the placement of the the various factors in this diagram isarbitrary; their exact position in the complex are not yet known. TFIIE, TFIIH, TFIIJ D Pol II PIC formation in order: B F TFIID→A →B →F →pol II →E →H Transcription starts 42

  43. 43

  44. 1.2 Elongation of mRNA transcription Elongation When the transcription initiation complex is formed, the transcription begins and the elongation of the RNA chain continues until termination occurs. 44

  45. AAAAA------AAAA 5’ 5’ AAUAAA RNA pol II 3’ 5’ GTGCGG TTATTT 5’ 3’ 1.3 Termination of mRNA transcription Unlike RNA polymerase in prokaryotes, RNA polymerase II does not terminate transcription at specific sites but rather transcription stops at varying distances downstream of the gene. Adding of poly A tail Termination site Cleavage site Modification signal 45

  46. Transcription termintation and adding of tail at 3’-end in eukaryotes 46

  47. 5’ 3’ 2. Synthesis of rRNA in Eukaryotes rRNA is synthesized in nucleolus. Each transcriptional unit contains sequence for 18S, 5.8S, and 28S rRNA. Several hundred copies of transcriptional units occur tandem in chromosome. Transcriptional units are separated by gene spacer sequences(基因间隔序列） DNA Transcription Pre-RNA 5.8S rRNA 28S rRNA 18S rRNA Class I promoter and RNA pol I are required 47

  48. Box A Box B Intermediate element Box A Box C 3. Synthesis of tRNA and 5S rRNA in Eukaryotes RNA pol III and TF IIIs are required One unusual feature of RNA pol III action is the location of TF III binding site within DNA sequence called as “internal control region”(内部控制区） The internal promoter of tRNA gene is split into two parts: box A and box B but those of 5S rRNA gene split into three parts: box A, a short intermediate element, box C tRNA gene 5S rRNA gene 48

  49. Questions What are requirements for mRNA biosynthesis in eukaryotes ? Specially for initiation stage? 49

  50. Questions 1. A promoter is A. a manager for a sports team. B. a specific sequence of DNA to which RNA polymerase binds. C. a specific sequence of DNA to which a catabolic repressor binds. D. a specific DNA sequence to which a restriction endonuclease binds. E. not found in eukaryotic cells. 50

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